文摘
In the present report, we describe a β galactosidase release (BGR) assay to evaluate cytotoxic T lymphocyte (CTL) activity against specific targets. Transient expression of β galactosidase (β gal) was obtained by infection with recombinant β gal vaccinia virus. Incubation of target cells with effector cells resulted in the release of β gal depending on the infection time and the effector/target cell ratio. BGR was evaluated using the chemiluminescent substrate, AMPGD (3-{4-Methoxyspiro[1,2-dioxetane-3,2′-tricyclo(3.3.1.13,7)decan]-yl}phenyl-b--galactopyranoside), a phenylgalactose-substituted 1,2-dioxetane compound. The use of a digenic vector carrying two genes coding for the β gal gene and the antigen, respectively, permits expression of the two proteins in the same cell. Coinfection of target cells with two different vectors, carrying β gal and antigen genes, respectively, was demonstrated to be as efficient as digenic vector when using high multiplicity of infection (MOI). The BGR assay was compared to the standard 4 h 51chromium (51Cr) release assay both in mouse and human models and showed comparable sensitivity. The BGR assay, therefore, provides a simple, specific and responsive method for measuring cell-mediated cytotoxic activity.
